We propose to neurochemically elucidate two presynaptic processes involved in the regulation of cholinergic transmission. In one project, the calcium-dependent regulation of neuronal high affinity choline transport will be studied using proteoliposomes derived from rat cortical synaptic plasma membranes. These reconstituted proteoliposomes retain high affinity choline uptake, and their internal millieu is accessible to direct ionic and pharmacological manipulations. In addition, we will determine the optimal solubilizing and reconstituting conditions for the eventual purification of this cholinergic neuron-specific transporter. Another project investigates the modulation of ACh release by presynaptic muscarinic and purinergic receptors, using synaptosomes from the purely cholinergic Torpedo electroplax or from rat brain. Mechanistic studies involving muscarinic modulation focus on a tentative working model in which changes in calcium-uptake, cyclic GMP levels, and protein phosphorylation are measured, along with membrane potential and cation channel activity. A related side project involves the solubilization and partial isolation of the voltage-dependent calcium-channel, using (3H)-nitrendipine binding as a marker. The long-term goals of this research are to : 1) develop new techniques to alter cholinergic transmission presynaptically; 2) elucidate the neurochemical processes underlying neurotransmitter release; and 3) purify neuron-specific plasma membrane protein-markers.